Method of treating psychological and metabolic disorders using IGF or IGF/IGFBP-3

ABSTRACT

Methods are provided for treating or alleviating the symptoms of subjects with psychological disorders, metabolic disorders, chronic stress-related disorders, sleep disorders, conditions associated with sexual senescence, aging, or premature aging by treating such subjects with IGF or mutant IGF either alone or complexed with IGFBP-3. Methods for increasing the levels of DHEA or DHEAS and treating or alleviating the symptoms of subjects with disorders characterized by low levels of DHEA or DHEAS by administering effective amounts of IGF or mutant IGF alone or complexed with IGFBP-3 are also provided. Methods for increasing the level of T4 and treating or alleviating the symptoms of subjects with disorders characterized by low levels of T3 or T4 by administering effective amounts of IGF or mutant IGF alone or complexed with IGFBP-3 are additionally provided.

This application is a divisional of U.S. patent application Ser. No.09/088,618, filed Jun. 1, 1998 now U.S. Pat. No. 6,025,332, which is acontinuation-in-part of U.S. patent application Ser. No. 08/837,603,filed Apr. 21, 1997 now U.S. Pat. No. 6,015,786, which is acontinuation-in-part of U.S. patent application Ser. No. 08/805,807,filed Feb. 25, 1997 now U.S. Pat. No. 6,025,368, each of which isincorporated by reference its entirety.

FIELD OF THE INVENTION

The invention relates generally to the field of treating psychologicaland metabolic disorders, and relates particularly to the treatment ofthese disorders by administering insulin-like growth factor (IGF) aloneor complexed with insulin-like growth factor binding protein-3(IGFBP-3).

BACKGROUND OF THE INVENTION

DHEA

Dehydroepiandrosterone (DHEA) and its sulfated form,dehydroepiandrosterone-sulfate (DHEAS) are the principal circulatingsteroids in humans. These two steroids are synthesized in the adrenalcortex and are normally found at about a 1:1000 molar ratio in serum.DHEAS is thought to be the storage form of DHEA, and can be converted toDHEA by the action of a sulfatase. DHEA can serve as a substrate for theproduction of androgenic steroids, both in the steroidogenic organs(adrenal glands, gonads and placenta) and in peripheral tissues, such asthe skin, liver and brain.

DHEA is synthesized from pregnenolone in a two step reaction bycytochrome P450c17 (CYP17). CYP17 has both 17α-hydroxylase activity(which converts pregnenolone into 17α-hydroxypregnenolone, which is acortisol precursor) and 17,20-lyase activity (which converts17α-hydroxypregnenolone to DHEA). Purified CYP 17 has very low17,20-lyase activity. However, addition of cytochrome b5 enhances the17,20-lyase activity of cytochrome P450c17, resulting in increasedproduction of DHEA from pregnenolone and decreased production ofcortisol (Katagiri et al. (1995) Arch. Biock Biophys. 317(2):343-347).IGF-I has been reported to increase transcription of the CYP17 gene incultured Leydig cells, although expression of the 3β-hydroxysteroiddehydrogenase gene (which encodes an enzyme involved in the conversionof DHEA into androgenic steroids and 17α-hydroxypregnenolone intocortisol) was not affected. Insulin-like growth factor I (IGF-I) alsoincreases choriogonadotropin-stimulated production of testosterone byLeydig cells (Chuzel et al. (1996) Eur. J. Biochem. 239:8-16).

DHEA and DHEAS levels normally peak in the second or third decade oflife, declining by 80% or more of peak levels by age 70. Low levels ofDHEA and DHEAS are associated with a variety of disease conditions,including Alzheimer's Disease and cardiovascular disease. U.S. Pat. No.5,527,789 to Nyce suggests that high levels of DHEA (such as thosecaused by administration of DHEA or DHEAS) can cause cardiovasculardisease due to depletion of cardiac ubiquinone, but Aberg et al. ((1996)Chem. Biol.Interact. 99(1-3):205-218) shows that cardiac ubiquinonelevels are unaffected by DHEA administration.

DHEA and its derivatives have been described as treatments for a widevariety of conditions, including memory dysfunction, prostatichypertrophy, immune dysfunction, alopecia, for inhibiting plateletaggregation, and minor and major depression as well preventatives forcancer and cardiovascular disease (U.S. Pat. Nos. 4,835,147, 5,077,284,5,407,684, 5,162,198, 5,407,927, and 5,527,789 and International PatentApplication No. WO 94/6709). DHEA is also known to increase REM sleep inrats and humans, suggesting its utility for the treatment of sleepdisorders, memory loss and age-related dementia (Robel and Baulieu(1995) Ann. NY Acad. Sci. 774:82-110).

Administration of DHEA has been reported to increase serum levels ofIGF-I (U.S. Pat. No. 5,407,927; Morales et al. (1994) J. Clin.Endocrinol. Metab. 78(6):1366-1367) and to increase the sense ofwell-being, but not the libido, of subjects receiving DHEA. However,these reports do not establish any linkage between the elevation ofIGF-I levels and an improved sense of well-being.

Direct admninistration of DHEA, DHEAS and their derivatives can lead toserious side effects. For example, acne, hair loss, hirsutism anddeepening of the voice have been reported with use of DHEA in women. Inmen, excess DHEA may stimulate the growth of prostatic cancer. Thus,gratuitous addition of these steroid hormones individually to thecirculation has been shown to be complicated in practice. Directadministration of pharmacological amounts of DHEA and/or DHEAS may causea hormonal imbalance, which may in turn cause the side effectsassociated with DHEA and DHEAS administration.

Thyroid Hormones

The thyroid hormones, triiodothyronine (T3) and tetralodothyronine (T4)are major metabolic regulators in mammals. T4 is less active than T3,and can be converted to T3 in peripheral tissues. Administration of T4or T3 increases metabolism, erythropoiesis, bone turnover and the rateof muscle relaxation. Although thyroid hormones increase the rate ofprotein synthesis, hypertyoidism is associated with weight loss andmuscle wasting. Hypothyroidism can be accompanied by lethargia,decreased pulmonary function (hiypoventilation), low cardiac output anddecreased renal output. The thyroid hormones also interact with otherendocrine hormones, including the growth hormone axis and steroidalhormones.

T4 and T3 are synthesized from thyroglobulin, a protein that isiodinated on its tyrosine residues. Two iodinated tyrosines arecondensed to form a molecule of T4 or T3. Thyroglobulin, which is storedextracellularly in the follicular lumen of the thyroid gland, acts as astorage molecule for the iodinated tyrosine residues. Iodinated tyrosineresidues are released from thyroglobulin by intracellular proteolysis inthyroid cells. IGF-I has been shown to increase transcription ofthyroglobulin in FRTL-5 (rat thyroid) cells (Kamikubo et al. (1990) Mol.Endocrinol., 4:2021-2029). The influence of increased levels ofthyroglobulin mRNA on T4 and T3 levels is, however, unknown.

IGF

IGF-I and IGF-II are growth factors that have related amino acidsequence and structure, with each polypeptide having a molecular weightof approximately 7.5 kilodaltons (Kd). IGF-I mediates the major effectsof growth hormone, and thus is the primary mediator of growth afterbirth. IGF-I has also been implicated in the actions of various othergrowth factors, since treatment of cells with such growth factors leadsto increased production of IGF-I. In contrast, IGF-II is believed tohave a major role in fetal growth. Both IGF-I and IGF-II haveinsulin-like activities (hence their names), and are mitogenic(stimulate cell division) and/or are trophic (promote recovery/survival)for cells in neural, muscular, reproductive, skeletal and other tissues.

Unlike most growth factors, IGFs are present in substantial quantity inthe circulation, but only a very small fraction of this IGF is free inthe circulation or in other body fluids. Most circulating IGF is boundto the IGF-binding protein IGFBP-3. IGF-I may be measured in blood serumto diagnose abnormal growth-related conditions, e.g., pituitarygigantism, acromegaly, dwarfism, various growth hormone deficiencies,and the like. Although IGF-I is produced in many tissues, mostcirculating IGF-I is believed to be synthesized in the liver.

IGF is known to bind to at least three different cellular receptors; thetype 1 IGF receptor, the type 2 IGF receptor, and the insulin receptor(to which IGF binds with much lower affinity than the type 1 or 2receptor). Mutants of IGF-I have been described which have alteredbinding to one or more of these cellular receptors. Mutations at residue24 (normally tyrosine) to non-aromatic residues or replacement ofresidues 28-37 selectively affects binding to the type 1 receptor, whilemutations at residues 49-51 can selective reduce type 2 receptorbinding. Mutations at residue 60 (from tyrosine to non-aromatic aminoacids) can alter binding to the type 1 and 2 IGF receptors as well asthe insulin receptor (Cascieri et al. (1988) Biochemistry 27:3229-3233;Cascieri et al. (1989) J. Biol. Chem. 264:2199-2202; Bayne et al. (1988)J Biol. Chem. 264:11004-11008; Bayne et al. (1990) J. Biol. Chem.265:15648-15652).

Almost all IGF circulates in a non-covalently associated ternary complexcomposed of IGF-I or IGF-II, IGFBP-3, and a larger protein subunittermed the acid labile subunit (ALS). The IGF/IGFBP-3/ALS ternarycomplex is composed of equimolar amounts of each of the threecomponents. ALS has no direct IGF binding activity and appears to bindonly to the IGF/IGFBP-3 binary complex. The IGF/IGFBP-3/ALS ternarycomplex has a molecular weight of approximately 150 Kd. This ternarycomplex is thought to function in the circulation “as a reservoir and abuffer for IGF-I and IGF-II preventing rapid changes in theconcentration of free IGF” (Blum et al., pp. 381-393, Modern Concepts inInsulin-Like Growth Factors (E. M. Spencer, ed., Elsevier, New York,1991).

Some mutant IGF-Is exhibit altered binding to IGFBP-3 and/or alterationsin the ability to form the ternary complex. For example, mutations atresidues 3, 4, 8, 9, 12, 15, 16 and 24 (B domain mutants) and mutationsat residues 49-51 reduce formation of the binary complex, whilemutations involving residues 55 and 56, as well as IGF-I where residues63-70 (1-62 IGF-D) were deleted or residues 28-37 were replaced with aGly₄ linker (1-27-Gly₄-38-70 IGF-I) or where both changes were made(1-27-Gly₄-38-62 IGF-I)) and mutants thereof (for example, 1-62 IGF-Iwhere residue 24 was also changed) actually have increased binding toIGFBP-3. Some of these mutants, particularly the 1-62 IGF-I with amutation at residue 24, have a reduced capacity for formation of theternary complex, even though formation of the binary complex isincreased (Baxter et al. (1992) J. Biol. Chem. 267:60-65).

Nearly all of the IGF-I, IGF-II and IGFBP-3 in the circulation is incomplexed form, so very little free IGF is detected. Moreover, a highlevel of free IGF in blood is undesirable. High blood levels of free IGFwould lead to serious hypoglycemia due to the insulin-like activities ofIGF. In contrast to the IGFs and IGFBP-3, there is a substantial pool offree ALS in plasma which assures that IGF/IGFBP-3 complex entering thecirculation immediately forms the ternary complex.

IGFBP-3 is the most abundant IGF binding protein in the circulation, butat least five other distinct IGF binding proteins (IGFBPs) have beenidentified in various tissues and body fluids. Although these proteinsbind IGFs, they each originate from separate genes and have unique aminoacid sequences. Thus, the binding proteins are not merely analogs orderivatives of a common precursor. Unlike IGFBP-3, the other IGFBPs inthe circulation are not saturated with IGFs. Moreover, none of theIGFBPs other than IGFBP-3 can form the 150 Kd ternary complex.

IGF-I and IGFBP-3 may be purified from natural sources or produced byrecombinant means. For instance, purification of IGF-I from human serumis well known in the art (Rinderknecht et al. (1976) Proc. Natl. Acad.Sci. USA 73:2365-2369). Production of IGF-I by recombinant processes isshown in EP 0 128 733, published in December of 1984. IGFBP-3 may bepurified from natural sources using a process such as that shown inBaxter et al. (1986, Biochem. Biophys. Res. Comm. 139:1256-1261).Alternatively, IGFBP-3 may be synthesized by recombinantly as discussedin Sommer et al., pp. 715-728, Modern Concepts of Insulin-Like GrowthFactors (E. M. Spencer, ed., Elsevier, New York, 1991). RecombinantIGFBP-3 binds IGF-I in a 1:1 molar ratio.

Topical administration of IGF-I/IGFBP-3 complex to rat and pig wounds issignificantly more effective than administration of IGF-I alone (Id.).Subcutaneous administration of IGF-I/IGFBP-3 complex tohypophysectomized, ovariectomized, and normal rats, as well asintravenous administration to cynomolgus monkeys, “substantiallyprevents the hypoglycemic effects” of IGF-I administered alone (Id.).

IGF has been proposed as a treatment for a wide variety of indications.U.S. Pat. Nos. 5,434,134, 5,128,320, 4,988,675, 5,106,832, 5,534,493,5,202,119 and 5,273,961 and have disclosed the use of IGF for thetreatment of cardiomyopathy and myocardial infarction, steroid-inducedcatabolism, type II (insulin resistant) diabetes, renal disorders,pancreatic disorders, for increasing humoral immune response and forprevention of acute renal failure, respectively. Additionally, EuropeanPatents Nos. EP 434 625, EP 436469 and EP 560 723 and InternationalPatent Applications Nos. WO 93/23071, WO 91/12018, WO 92/00754, WO93/02695 and WO 93/08826 disclose the use of IGF for the treatment ofbone disorders, type I (juvenile or insulin-responsive) diabetes andgastrointestinal disorders.

The use of IGF complexed with IGFBP-3 has also been described for use inthe treatment of a variety of conditions. U.S. Pat. Nos. 5,200,509,5,187,151, 5,407,913, and 5,527,776 disclose the use of IGF/IGFBP-3complex for the treatment of osteoporosis, for inducing an anabolicstate when given by subcutaneous bolus injection, for increasing tissuerepair when given systemically, and for treating anemia. InternationalPatent Applications Nos. WO 95/03817, WO 95/08567, WO 95/13823, WO95/13824, WO 96/02565 disclose the use of IGF/IGFBP-3 complex for thetreatment of disorders of the reproductive, immunologic, neural, renal,and skeletal systems.

In addition to its activities in other organ systems, IGF has trophiceffects on the cells of the peripheral and central nervous system. IGF'strophic effects on neural cells include promoting the survival of avariety of neuronal cell types as well as promoting neurite outgrowth inmotor neurons. U.S. Pat. Nos. 5,093,317, 5,420,112, 5,068,224, andInternational Patent Applications Nos. WO 93/02695, WO 93/08826 and WO95/13823 describe the use of IGF or IGF complexed to IGFBP-3 for thetreatment of disorders of the nervous system, exploiting IGF's trophicactivity on the cells of nervous tissues. None of these patents orpublications disclose or suggest the use of IGF for the treatment ofpsychological disorders or memory loss.

Other disorders exist which would benefit from a reduction in the levelsof IGF. For example, some cancer cells are dependent on IGF forcontinued survival (Resnicoffet al. (1994) Cancer Res. 54:2218-2222).Reduction in circulating IGF levels could result in tumor progression,as IGF-dependent tumor cells undergo apoptosis. In autoimmune disorders,reduction in IGF levels could reduce symptoms of these disorders, as IGFis known to have stimulatory effects on the immune system.

None of the references disclosed above disclose or suggest the use ofIGF or IGF/IGFBP-3 complex for the treating or alleviating the symptomsof psychological or metabolic disorders. Further, none of the citedreferences disclose or suggest the administration of IGF or IGF/IGFBP-3complex for treating or alleviating the symptoms of sleep disorders orfor treating or alleviating symptoms and disorders associated withsexual senescence.

Psychological Disorders

The acuity of memory gradually declines with age, and can also beaffected by a variety of disorders. Memory can be characterized invarious ways, including declarative or explicit (involving recall andrecognition) versus implicit (involving skills and conditioning). Anumber of compounds have been suggested as treatments for enhancingmemory, including cholinergic agonists and cholinesterase inhibitors,calcium channel blockers, angiotensin convertring enzyme(ACE)-inhibitors such as captopril and peptides such as vassopressin andcorticotropin (which induce the synthesis of adrenal steroids), andothers (Mondadori et al. (1994) Proc. Natl. Acad. Sci. USA 91:2041-2045). Steroid hormones have also been used to treat-memory loss.Administration of pregnenolone sulfate (PS), dehydroepiandrosteronesulfate (DHEAS), androstenedione (A), testosterone and aldosterone(among others) were effective in improving retention in a rodent model(Flood et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1567-1571). PS andDHEAS were active as memory enhancers when injected into thehippocampus. PS was also active when injected into the amygdala andmammillary bodies but not the caudate nucleus (Flood et al. (1995) Proc.Natl. Acad. Sci. USA 92: 10806-10810). Pregnenolone and DHEA arebelieved to act in a paracrine fashion at neurons, thus modifying sleepEEG in humans in a manner that suggests their potential as memoryenhancers (Holsboer et al. (1994) Ann. NY Acad. Sci. 746: 345-361).

IGF-I has been shown to induce long term depression of glutamate-inducedgamma-aminobutyric acid release in the cerebellum (Castro-Alemancos etal. (1993) Proc. Natl. Acad. Sci. USA 90: 7386-7390). This finding ledCastro-Alemancos et al. to test the effects of IGF-I on motor learningand retention (i.e., implicit memory), using the “eye-blink response” asthe indicator. Experimental results led them to postulate that IGF-Iplays a role in learning the eye-blink response but they found noevidence for a role for IGF-I in retention or memory (Castro-Alemancoset al. (1994) Proc. Natl. Acad. Sci. USA 91: 10203-10207).

There is a need in the art for an effective method for enhancing memoryand for treating and/or alleviating the symptoms of memory loss.

Depressive disorders are common among the population. Seventeen percentof the population is expected to suffer from major depression-duringtheir lifetime prevalence (Andrews et al. (1994) Am. J. Med.97:24S-32S). Approximately two-thirds of patients respond toantidepressant medication. Nevertheless, all effective classes ofantidepressants have significant side-effect profiles. Selectiveserotonin reuptake inhibitors (SSRIs) cause nausea, headache and sexualdysfunction. Additionally, many SSRIs inhibit a number of hepaticcytochrome P450 isozymes which can lead to serious drug interactionproblems in addition to the side effects caused directly by the drugsthemselves. Tricyclics are cardiotoxic and overdoses are frequentlyfatal. Other classes can cause seizures, priapism and elevations inblood pressure (Andrews et al. (1994) Am. J. Med. 97: 24S-32S).

Primary hypothyroidism is a relatively common endocrine disorder thatdevelops insidiously and can mimic depression. Between and 8 and 14percent of patients diagnosed with depression have some degree ofhypothyroidism (Tallis (1993) Brit. J. Clin. Psychol. 32:261-270).Primary hypothyroidism may be treated by increasing levels of T4 and/orT3.

There is a need in the art for a method for treating and/or alleviatingthe symptoms of depression of multiple etiologies.

Metabolic Disorders

Spinal chord injury (SCI) in adult males may result in various hormonaland metabolic abnormalities—both as a result of injury and secondary toreduced exercise and mobility. Various studies in this patientpopulation have documented the following abnormalixties in subjects withSCI relative to the normal population: a suppression in the GH responseto growth hormone releasing hormone (GHRH), arginine or other agents;significantly lower IGF-I levels; elevated follicle stimulating hormone(FSH) and leutinizing hormone (LH) levels; reduced thyroid hormonelevels; hyperprolactinemia (in quadriplegics only); hypogonadismassociated with lowered testosterone levels; increased frequency ofurinary tract infections; obesity; high prevalence of carbohydrateintolerance; diabetes; low HDL cholesterol; lowered cardiopulmonaryfitness and dyspnea at rest; increased susceptibility to coronary arterydisease; deficient bowel control (esp. constipation); lower restingmetabolic rate; and active pressure sores (Shetty et al. (1993) Am J.Med Sci. 305:95-100; Huang et al. (1995) Metabolism 44:1116-20;Tsitouras et al. (1995) Horm. Metab. Res. 27:287-292; Geders et al.(1995) Am. J. Gastroenterol 90:285-289; Bauman et al. (1994) Metabolism43:749-756; Bauman et al. (1994) J. Clin. Endocrinol. Metab.78:1135-1138; Almenoff et al. (1995) Paraplegia 33:274-277; Bauman etal. (1994) Horm. Metab. Res. 26:152-156; Kahn et al. (1996)Proc.Natl.Acad.Sci. USA 93:245-249).

Atelectasis (insufficient lung inflation/deflation) and pneumonia arethe major causes of morbidity and mortality in patients with SCI (citedin Almenoff et al., supra). In a study of 165 SCI subjects, forced vitalcapacity and other measures of pulmonary function were inverselycorrelated with with the level of injury (i.e., the higher the level ofinjury, the lower the parameter; Almenoff et al. (1995) Lung173:297-306). Other studies have shown that thyroid hormone (both T3 andT4) levels are significantly lower in quadriplegics than in paraplegicsor normal subjects (Huang et al., supra). Thyroid hormone levels arecorrelated with pulmonary function. Further, hypothyroid individuals areknown to suffer from breathing difficulties.

Most of the metabolic abnormalities experienced by SCI victims are alsoincreasingly observed during the normal aging process in humans. Forthis reason, SCI may provide an excellent model for the study of thenormal aging process as well as premature aging (Bauman et al. (1994)Horm. Metab. Res., supra). There are also several genetic diseases whichcause premature aging, including ataxia telangiectasia, Werner'ssyndrome, Hutchinson-Guilford progeria, and Cockayne's syndrome. It isexpected that symptoms that are shared between SCI, aging and prematureaging would benefit from the same treatment.

Accordingly, there is a need in the art for an effective treatment foralleviating symptoms associated with SCI and for the symptoms of agingand premature aging.

Stress hormones can profoundly affect the workings of all endocrinesubsystems, resulting in a condition referred to ashypothalamic-pituitary axis dysregulation (HPA dysregulation). Forexample, individuals under stress do not experience the normal increasein growth hormone levels following induction of hypoglycemia (induced byadministration of insulin); instead, IGF-I levels drop and cortisol andnorepinephrine levels rise (Ferraccioli et al. (1994) J. Rheumatol.21:1332-1334). This abnormal response is believed to play a role inlowering IGF-I levels in chronically stressed conditions such asfibromyalgia (Id.).

Fibromyalgia is a relatively common disorder with a prevalence in thegeneral population of between 2 and 4% (Wolfe et al. (1990) ArthritisRheum. 33:160-172). Fibromyalgia, which is approximately twice as commonin women as in men, is characterized by widespread pain, tenderness,fatigue, sleep disturbance, paresthesias, anxiety, and other similarsymptoms. Fibromyalgia has many symptoms in common with chronic fatiguesyndrome and the two conditions are frequently treated with the samedrugs. Interestingly, while sleep disturbances are associated withfibromyalgia, sleep deprivation itself can induce the symptoms offibromyalgia (Moldofsky et al. (1975) Psychosom. Med. 37:341-351).

Physical and psychological stressors elevate plasma levels of IL-6. Thesource of this IL-6 is unknown, but it has been shown to be non-immune(Zhou et al. (1993) Endocrinol. 133:2523-2530). DHEAS has been shown toreduce chronically elevated levels of IL-6 (Daynes et al. (1993) J.Immunol. 150:5219-5230).

HPA dysregulation is also observed when sleep patterns are disrupted.Melatonin inhibits the basal and stimulated release of hypothalamicvassopressin in vitro (Yasin et al. (1993) Endocrinol. 132:1329-1336),and sleep inhibits activation of adrenocorticotropic hormone (ACTH) andcortisol secretion. Conversely, through the mineralocorticoid (slow wavesleep) and glucocorticoid (rapid eye movement—REM—sleep) receptors,cortisol can exert feedback effects on sleep patterns (Holsboer et al.(1994) Ann. N.Y. Acad. Sci. 746:345-361). DHEA affects the duration ofREM sleep and this may explain some of its actions on the consolidationof memory.

There is a need in the art for effective methods for treating disordersassociated with chronic stress and/or alleviating the symptoms ofdisorders associated with chronic stress.

The thalassemias are a group of genetic diseases which share the symptomof reduced levels of hemoglobin in the blood (i.e., anemia). This is dueto reduced or absent production of either adult alpha or adult betahemoglobin. The existence of thalassemia in a subject emerges during thefirst month of life, as fetal hemoglobin is down regulated and adulthemoglobin is up regulated. Thalassemic individuals are unable to switchover to adult hemoglobin and become anemic and dependent ontransfusions. In addition to anemia, thalassemia patients alsoexperience symptoms relating to high levels of toxic products such asiron and billrubin. Animal models, including naturally occurring mutantsand introduced mutants, are available for testing treatments for betathalassemias (Popp et al. (1984) Proc. NY. Acad. Sci. 445:432-444;Ciavatta et al. (1995) Proc. Natl. Acad Sci. USA 92:9259-9263; Yang etal. (1995) Proc. Natl. Acad. Sci. USA 92:11608-11612). A need exists inthe art for effective methods for improving or alleviating symptomsassociated with deficiencies of detoxification in thalassemia patients.

Sexual Senescence

The hypothalamic-pituitary-gonadotropic axis is responsible for theproper function of reproductive organs as well as for some aspects ofreproductive behavior. Although IGF-I has been implicated in gameteformation, little is known about its effects on sexual behavior. IGF-Ihas been implicated in the gonadotropic axis in a study of Igr1knock-out mice, which are infertile dwarfs with drastically reducedlevels of serum testosterone (Baker et al. (1996) Mol. Endocrinol.10:903-918).

Androgen levels are known to decrease with aging in men. Androgendeficiency in men has been linked to decreased muscle mass, asthenia,osteoporosis and decreased sexual activity and, in some cases, changesin mood and cognitive function. In women, studies have reported arelationship between the transition into menopause and a decline insexual interest and activity as measured by a variety of symptoms.Estrogen can affect some (but not all) of these symptoms Nathorst-Booset al. (1993) Acta Obstet. Gynecol. Scand. 72:656-660). In one study,McCoy Sexual Rating score (which relates to parameters such as thefrequency of sexual fantasies, impaired lubrication and pleasure fromintercourse) correlated with levels of circulating IGF-I (Nathorst-Booset al. (1993) J. Psychosom. Obstet. Gynaecol. 14:283-293).

DHEA and its derivatives have been suggested as treatments for somesymptoms of sexual senescence, such as prostatic hypertrophy and sexualdysfunction associated with menopause. U.S. Pat. No. 4,835,147 teachesthe administration of DHEA for the treatment of prostatic hypertrophyand sexual dysfunction symptoms related to nervous system dysfunction.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “IGF” refers to insulin-like growth factor from anyspecies. IGF includes both IGF-I and IGF-II, in native-sequence orvariant form, including but not limited to naturally-occurring allelicvariants. IGF may be from any source, whether natural, synthetic orrecombinant.

As used herein, “IGF-1” refers to insulin-like growth factor I from anyspecies, including bovine, ovine, porcine and human, in native-sequenceor variant form, including but not limited to naturally-occurringallelic variants. IGF-I may be from any source, whether natural,synthetic or recombinant, provided that it will bind IGFBP-3 at theappropriate site. Preferred herein is human native-sequence, matureIGF-I, preferably without an amino-terminal methionine. More preferably,the native sequence, mature IGF-I is produced recombinantly, forexample, as described in PCT publication WO 95/04076.

As used herein, the term “mutant IGF-I” refers to which have alteredamino acid sequences at one or more sites in the molecule. Mutant IGF-Iretains its ability to bind IGFBP-3, but may be altered in its otherproperties, such as binding to the type I or type II IGF receptor orbinding to the insulin receptor. Descriptions of mutant IGF-Is may befound in Cascieri et al. (1988) Biochemistry 27:3229-3233; (1989) J.Biol. Chem. 264:2199-2202), Bayne et al. (1990) J. Biol. Chern.265:15648-15652) and Baxter et al. (1992) J. Biol. Chem. 267:60-65).Examples of mutant IGF-I include mutants in which one or more of IGF-Istyrosine residues (i.e., residues 24, 31, or 60) are replaced withnon-aromatic residues (i.e., other than tyrosine, phenylalanine ortryptophan), mutants where amino acid residues 49, 50, 51, 53, 55 and 56are altered (for example, where residues 49-50 are altered toThr-Ser-Ile or where residues 55-56 are altered to Tyr-Gln).

As used herein, “IGF-II” refers to insulin-like growth factor II fromany species, including bovine, ovine, porcine and human, innative-sequence or variant form, including but not limited tonaturally-occurring allelic variants. IGF-II may be from any source,whether natural, synthetic or recombinant, provided that it will bindIGFBP-3 at the appropriate site. Preferred herein is humannative-sequence, mature IGF-II, preferably without an amino-terminalmethionine. More preferably, the native sequence, mature IGF-I isproduced recombinantly, for example, as described in PCT publication WO95/04076.

As used herein, “IGFBP-3” refers to insulin-like growth factor bindingprotein 3. IGFBP-3 is a member of the insulin-like growth factor bindingprotein family. IGFBP-3 may be from any species, including bovine,ovine, porcine and human, in native-sequence or variant form, includingbut not limited to naturally-occurring allelic variants. PreferedIGFBP-3 embodiments include native sequence human IGFBP-3 and variantsof human IGFBP-3 wherein the one or more of the asparagine residueswhich form the normal N-linked glycosylation sites (positions 89, 109and 172) are changed to aspartate (e.g.: N89D; N109D; N172D; N89D,N109D;N89D,N172D; N109D,N172D; and N89D,N109D,N172D variants) or to otheramino acid residues (e.g.: N89X; N109X; N172X; N89X,N109X; N89X,N172X;N109X,N172X; and N89X,N109X,N172X variants) as well as variants whichhave been altered to improve resistance to degradation, such asalterations at positions 116 and 135 (e.g, D116E, D135E andD116E,D135E), or alterations which affect the nuclear localizationsignal (NLS) of IGFBP-3, which is located at residues 215 through 232(Radulescu, 1994, Trends Biochem Sci. 19(7):278). Examples of preferredNLS variant IGFBP-3s include K228E, R230G, K228E, R230G, K228X, R230X,and K228X, R230X, as well as variations at residues 215, 216 and 231. Ofcourse, a variant IGFBP-3 may include more than one type of variation(e.g., a variant IGFBP-3 may be both ND variant and degradationresistant variant). IGFBP-3 can form a binary complex with IGF, and aternary complex with IGF and the acid labile subunit (ALS). IGFBP-3 maybe from any source, whether natural, synthetic or recombinant, providedthat it will bind IGF-I and ALS at the appropriate sites. Preferably,IGFBP-3 is produced recombinantly, as described in PCT publication WO95/04076.

A “therapeutic composition,” as used herein, is defined as comprisingIGF-I complexed with its binding protein, IGFBP-3 (IGF-I/IGFBP-3complex). The therapeutic composition may also contain other substancessuch as water, minerals, carriers such as proteins, and other excipientsknown to one skilled in the art.

The term “metabolic disorder,” as used herein, is defined as disordersassociated with deleterious alterations in metabolism. Metabolicdisorders include, for example, hypothyroidism, disorders associatedwith chronic stress, symptoms associated with spinal cord injury such asdecreased pulmonary function, increased colonic transit time and thelike.

The term “psychological disorder,” as used herein, includes, but is notlimited to, disorders of mood and affect, memory dysfunction, motor andtic disorders, substance abuse disorders, psychotic disorders, andanxiety disorders. Psychological disorders may be recognized asdescribed in the Diagnostic and Statistical Manual of Mental Disorders:DSM-IV (American Psychiatric Assn., Washington, D.C., 4th ed., 1994)(“DSM-IV”).

Disorders of mood and affect include, for example, minor and majordepression, dysthymic disorder, bipolar disorders and the like. Mooddisorders are characterized in DSM-IV, and may be recognized by symptomssuch as decreased energy, insomnia, weight loss, depressed mood, loss ofpleasure or interest in most or all activities.

Memory dysfunctions are also referred to as amnestic disorders. Amnesticdisorders are generally characterized by an inability to learn newinformation or to recall previously learned information. Memorydysfunctions may be the result of pathological processes (e.g., trauma,hypoxia, disease) or may be a result of the normal aging process.

Motor and tic disorders are characterized by motor and/or vocal tics,and include, but are not limited to, Tourette's disorder, chronic motoror vocal tic disorder, transient tic disorder, and stereotypic movementdisorder. Tics are sudden, rapid, recurrent, nonrhythmic, stereotypedmovements (motor tics) or vocalizations (vocal tics). Motor tics includeeye blinking, shoulder movements such as shrugging, and facialgrimacing. Vocal tics include grunts, clicks, yelps, barks, snorts,coprolalia (use of socially unacceptable words) and echolalia (repeatingthe last heard sound).

Substance abuse disorders include disorders such as substancedependence, substance abuse and the sequalae of substanceabuse/dependence, such as substance-induced psychological disorders,substance withdrawal and substance-induced dementia or amnesticdisorders. Substance abuse disorders may be recognized by impairedcontrol of use of a substance such as alcohol, stimulants or narcotics,and may be accompanied by guilt or regret about use and failed attemptsto reduce use.

Psychotic disorders include such conditions as schizophrenia,schizofreniform disorder, schizoaffective disorder, and delusionaldisorder. Symptoms of psychotic disorders may include, but are notlimited to, delusions, hallucinations, disorganized speech (in which thepatient may “slip” from one topic to another, give answers to questionsthat are obliquely related or not at all related to the question, orhave speech so severely disorganized as to be incomprehensible), grosslydisorganized behavior (that may be manifested by unpredictableagitation, difficulty in maintaining hygiene, inappropriate clothing,inappropriate sexual behavior, etc.), negative symptoms (including lossor flattening of affect, avolition, anhedonia), and catatonic

Anxiety disorders are characterized by exessive anxiety, worry, fear,tension, or arousal that cause distress and/or a clinically significantdecrease in function. Anxiety disorders include, but are not limited to,panic disorder, phobias (including agoraphobia), obsessive-compulsivedisorder, and posttraumatic stress disorder.

“Sexual dysfunctions,” as used herein, refer to disorders ordysfunctions of sexual drive, sexual excitement and orgasm, as well asdysfunctions of male and female arousal, such as erectile andlubrication dysfunctions.

A “low level of circulating sex steroid,” as used herein, refers to aserum level of a sex steroid that is in the 30^(th) or lower percentilefor the population of the same species, sex, and approximate age (ie.,±5 years).

“Fibromyalgia,” as used herein refers to a syndrome that has beendefined by the American College of Rheumatology (Wolfe et al. (1990),supra). Fibromyalgia is characterized by widespread pain of at leastthree months duration that is present in the axial skeleton as well asall four quadrants of the body. Pain is also elicited at at least 11 of18 stereotypical pressure points.

Modes of Carrying Out the Invention

The inventors have unexpectedly found that administration of IGF/IGFBP-3complex can increase serum levels of DHEAS, T4, estrogen, andandrostenedione. While not wishing to be bound by any particular theory,the inventors propose that any disorder characterized by a deficiency ofDHEA, DHEAS, thyroid hormone or sex steroids and disorders that may betreated by administration of DHEA, DHEAS thyroid hormone or sex steroidsmay be treated by administration of IGF, preferably IGF complexed withIGFBP-3. Symptoms of disorders characterized by a deficiency of DHEA,DHEAS, thyroid hormone or sex steroids and disorders that may bealleviated by administration of DHEA, DHEAS thyroid hormone or sexsteroids may be treated by administration of IGF, preferably IGFcomplexed with IGFBP-3.

One embodiment of the invention is a method for increasing levels ofDHEAS in an individual, by administering IGF or IGF/IGFBP-3 complex.Administration of IGF or IGF/IGFBP-3 complex increases serum levels ofDHEAS.

In another embodiment, the invention relates to the use of IGF for thetreatment of psychological and metabolic disorders. Psychologicaldisorders include amnestic disorders such as memory dysfunction,disorders of mood and affect including mild and major depression, motorand tic disorders such as Tourette's disorder, substance abuse disordersincluding substance abuse and substance dependence, psychotic disorderssuch as schizophrenia, and anxiety disorders including posttraumaticstress disorder. Administration of IGF, preferably IGF/IGFBP-3 complexresults in improvements or alleviation of the symptoms of psychologicaldisorders.

IGF or IGF/IGFBP-3 complex may be administered for the treatment ofamnestic disorders, such as memory loss, particularly declarative memoryloss. Declarative memory (also known as explicit memory) involves recalland recognition, as opposed to implicit memory, which relates to memoryof skills and conditioning. Memory loss is associated with normal aging,as well as trauma, hypoxia and disease. Administration of IGF orIGF/IGFBP-3 complex improves memory function and alleviates the symptomsof amnestic disorders.

IGF or IGF/IGFBP-3 complex may be administered for the treatmentdisorders of mood and affect. IGF or IGF/IGFBP-3 complex treatmentalleviates or reduces the symptoms of such mood and affect disorders asmild depression, major depression, cyclothymic disorder, dysthymicdisorder, and bipolar disorder. Alleviation or reduction of symptoms ofdisorders of mood and affect may be indicated by improved mood,increased interest in any or all activities, reduction of feelings ofguilt, or other changes as will be apparent to one of skill in the art.

In another embodiment, the invention relates to the treatment ofdisorders of metabolism. The symptoms of disorders of metabolism such asprimary hypothyroidism, HPA axis dysregulation, and symptoms associatedwith spinal cord injury (SCI) are improved or alleviated byadministration of IGF or IGF/IGFBP-3. Improvements in the symptoms ofdisorders of metabolism may include decreased lethargy, reduced coldsensitivity, improved pulmonary function, decreased colonic transittime, and other measures which will be apparent to the skilled artisan.

The invention also relates to to the treatment of disorders which resultin premature aging, such as ataxia telangiectasia, Werner's syndrome,Hutchinson-Guilford progeria, and Cockayne's syndrome. Theadministration of IGF or IGF/IGFBP-3 results in improvements in thesymptoms of premature aging.

In a further embodiment, the invention relates to the treatment ofchronic stress-related conditions. Chronic stress-related conditionsinclude fibromyalgia, chronic fatigue syndrome, hypothalamic-pituitaryaxis dysregulation, chronic sleep deprivation, and conditions associatedwith elevated levels of interleukin 6 (IL-6). Administration of IGF orIGF/IGFBP-3 complex results in reduction or alleviation of the symptomsof chronic stress-related conditions.

One embodiment relates to the use of IGF or IGF/IGFBP-3 for thetreatment of sleep disorders. Administration of IGF or IGF/IGFBP-3improves the symptoms of sleep disorders, as measured by an increase inREM sleep.

Another embodiment of the invention relates to the administration of IGFor IGFBP-3 for the treatment of the symptoms of sexual senescence.Administration of IGF or IGF/IGFBP-3 results in improvements in thesymptoms of sexual senescence, such as a reduction in the symptoms ofprostatic hypertrophy and improvement in sexual dysfunctions.

Administration of IGF or IGF/IGFBP-3 results in increases in circulatinglevels of sex hormones, such as estradiol and androstenedione.Accordingly, administration of IGF or IGF/IGFBP-3 is useful for thetreatment of symptoms, disorders, and conditions associated with lowcirculating levels of sex steroids.

The invention also relates to increasing the detoxification capacity ofa subject. The administration of IGF or IGF/IGFBP-3 is useful forincreasing levels of cytochrome b5, a protein that plays important rolesin the modulation and regulation of cytochrome P450 pathways. Asdemonstrated in Example 2, administration of IGF-I/IGFBP-3 complexincreases serum levels of DHEAS without increasing levels of cortisol,indicating an increase in cytochrome b5 activity. Example 5 furthershows that administration of IGF-I/IGFBP-3 complex increases levels ofcytochrome b5 MRNA levels in blood cells. In addition to its activity instimulating DHEA synthesis, cytochrome b5 regulates the detoxificationfunctions of cytochrome P450. Therefore, administration of IGF orIGF-I/IGFBP-3 is useful for treating disorders associated withinsufficient detoxification activity, such as the thalassemias andalchohol or other drug-related toxicities. Administration of IGF orIGFBP-3 results in improvement or alleviation of symptoms of thalassemiaassociated with accumulation of toxic products such as iron andbilirubin.

The invention also provides for new methods for the reduction of IGFactivity in individuals in need of such reduction. As is shown inExample 2, administration of IGF-I/IGFBP-3 complex results in markedreductions in the level of IGF-II, accompanied by an increase in IGF-I.While not wishing to be bound to any one theory, the increased IGF-Ilevels are believed to be due to the IGF-I which has been administeredto the subjects. This reduction in IGF-II levels can be exploited toreduced overall IGF activity in the circulation, by adiministering acomplex of IGFBP-3 and a mutant IGF-I which has been engineered to havereduced binding to one or more of the IGF receptors (the type 1 and 2IGF receptors and the insulin receptor), while retaining the ability tobind IGFBP-3 and form the ternary complex. Administration of a complexof this mutant IGF-I and IGFBP-3 results in a reduction in the level ofactive IGF in the circulation, thereby reducing the numbers and/oractivity of cells and tissues which depend on IGF for survival oractivity.

In one embodiment, mutant IGF/IGFBP-3 complex is administered tosubjects having cancers dependent on IGF. Reduction of levels of activeIGF is useful in the treatment of cancers where the cancer cells areIGF-dependent for survival. Reduction of active IGF levels results inapoptosis of the IGF-dependent cancer cells, resulting in reduction intumor mass.

In another embodiment, mutant IGF/IGFBP-3 complex is administered tosubjects having autoimmune disorders. Autoimmune disorders such assystemic lupus erythematosis (“lupus” or “SLE”), in multiple sclerosis(“MS”), Grave's disease, Hashimoto's thyroiditis, Goodpasture'ssyndrome, myasthenia gravis (“MG”), insulin resistance, and otherdisorders known in the art to involve autoimmune reactions, will benefitfrom the administration of mutant IGF-I complexed to IGFBP-3. Immuneeffector cells are known to be stimulated by IGF. Accordingly, reductionof active IGF levels will reduce the stimulation of the immune cells,resulting in improvement or alleviation of the symptoms of autoimmunedisorders.

In a further embodiment, mutant IGF/IGFBP-3 complex is administered tosubjects having hyperthyroid conditions (i.e., excess levels of thyroidhormones). Hyperthyroid conditions will also benefit from theadministration of mutant IGF-I/IGFBP-3 complex. As shown herein, theadministration of IGF-I/IGFBP-3 increases levels of T4, and is useful inthe treatment of hypothyroidism. Reducing circulating levels of IGF willdecrease thyroid hormone levels, resulting in improvement and/oralleviation of the symptoms of hyperthyroid and other conditions whereit is desirable to reduce levels of thyroid hormone.

Another embodiment involves the administration of mutant IGF/IGFBP-3complex to subjects having conditions associated with excess levels ofandrogen hormones. Conditions involving an excess of androgen hormones,such as virilization, hirsutism, and other disorders known by one ofskill in the art to involve elevated levels of androgen hormones, willbenefit from the administration of mutant IGF-I/IGFBP-3 complex. Asshown herein, the administration of IGF-I/IGFBP-3 complex results inincreased levels of androgen hormones and in DHEAS, an androgen hormoneprecursor. Reduction of circulating IGF activity will decrease levels ofandrogen hormones, thereby improving or alleviating the symptoms ofdisorders involving excess androgen hormones or disorders where areduction in androgen hormones is beneficial.

In a further embodiment, mutant IGF/IGFBP-3 complex is administered tosubjects having hypophosphatemia (decreased serum concentrations ofphosphorus). Subjects having low serum phosphorus will benefit from theadministration of mutant IGF/IGFBP-3 complex. Administration of mutantIGF/IGFBP-3 complex results in increased serum phosphorus levels andimprovement or alleviation of the symptoms associated withhypophosphatemia.

The inventive methods disclosed herein provide for the parenteraladministration of IGF or IGF/IGFBP-3 complex to subjects in need of suchtreatment. Parenteral administration includes, but is not limited to,intravenous (IV), intramuscular (IM), subcutaneous (SC), intraperitoneal(IP), intranasal, and inhalant routes. IV, IM, SC, and IP administrationmay be by bolus or infusion, and in the case of SC, may also be by slowrelease implantable device, including, but not limited to pumps, slowrelease formulations, and mechanical devices. The formulation, route andmethod of administration, and dosage will depend on the disorder to betreated and the medical history of the patient. In general, a dose thatis administered by subcutaneous injection will be greater than thetherapeutically-equivalent dose given intravenously or intramuscularly.Preferably, the dose of IGF administered will be from about 25 μg/kg toabout 2 mg/kg of body weight. More preferably, the dose of IGF will befrom about 50 μg/kg to about 1 mg/kg. Most preferably the dose of IGFwill be from about 100 μg/kg to about 400 μg/kg.

The IGF is preferably IGF-I. A composition comprising equimolar amountsof IGF-I and IGFBP-3 is preferred. Preferably the IGF-I and IGFBP-3 arecomplexed prior to administration. Preferably, the complex is formed bymixing approximately equimolar amounts of IGF-I and IGFBP-3 dissolved inphysiologically compatible carriers such as normal saline, or phosphatebuffered saline solution. More preferably, a concentrated solution ofrhIGF-I and a concentrated solution of rhIGFBP-3 are mixed together fora sufficient time to form an equimolar complex. Most preferably, rhIGF-Iand rhIGFBP-3 are combined to form a complex during purification, asdescribed in International Patent Application No. WO 96/40736.

Mutant IGF-I is preferably a mutant IGF-I that retains binding toIGFBP-3, but has reduced binding to one or more of the cellularreceptors to which IGF-I normally binds. Preferred mutant IGF-Is thathave reduced binding to all IGF cellular receptors but that retainIGFBP-3 binding include, for example: mutant IGF-Is where position 60 isaltered; mutant IGF-Is where position 60 and other positions arealtered, such as positions 24, 31, 55 and 56. Preferred mutant IGF-Isthat have reduced type 1 IGF receptor binding but retain IGFBP-3 bindinginclude mutant IGF-Is with changes at position 24 and 31. Preferredmutant IGF-Is that have reduced type 2 IGF receptor binding but retainIGFBP-3 binding include mutations at positions 41, 45, and 46.

For parenteral administration, compositions of the complex may besemi-solid or liquid preparations, such as liquids, suspensions, and thelike. Physiologically compatible carriers include, but are not limitedto, normal saline, serum albumin, 5% dextrose, plasma preparations, andother protein-containing solutions. Optionally, the carrier may alsoinclude detergents or surfactants.

EXAMPLES Example 1

rhIGF-I and rhIGF-I/IGFBP-3 complex were administered to ovariectomizedmature rats, and the effects on an endocrine organ, the adrenal gland,were measured.

16 week old female Sprague-Dawley rats were obtained from Charles-RiversLaboratories and allowed to acclimate at least one week prior toovariectomy. Animals were housed separately in accordance with NIHguidelines, and allowed ad libitum access to Purina® brand RatLaboratory Chow 5001 and water. Animals were ovariectomized bilaterally,using a dorsal approach. Following ovariectomy, animals were allowed aneight week recovery period, then randomly assigned to the treatmentgroups shown in Table 1. rhIGF-I and rhIGF-IGFBP-3 complex (produced asdescribed in Sommer et al., supra) diluted in phosphate buffered saline(20 mM sodium phosphate, pH 6.0, 150 mM NaCl in pyrogen-free water) wereadministered to the test animals by daily subcutaneous injection.Control animals received daily subcutaneous injections ofphosphate-buffered saline. Animals were euthanized immediately followingthe completion of eight weeks of treatment.

Adrenal weights and body weights were measured for all animal groups atsacrifice. Relative adrenal weights were calculated by dividing theadrenal weight (mg) by the body weight (kg) for each animal. The resultsare shown in Table 1.

TABLE 1 TREATMENT n MEAN ± SD p. value* OVX Controls 11 132.7 ± 36.9 —IGF-I (0.9 mg/Kg)  7 147.1 ± 22.8 0.3216 IGF-I (2.6 mg/Kg)  7 161.4 ±47.8 0.2043 IGF-I groups combined 14 154.3 ± 36.7 0.1607 IGF-I complex(0.9 mg/Kg**)  8 172.5 ± 36.2 0.0327 IGF-I complex (2.6 mg/Kg**)  8160.0 ± 22.7 0.0634 IGF-I complex gps combined 16 166.3 ± 29.9 0.0219*versus OVX control group; two-tailed, unpaired t-test; **equivalentIGF-I dose present in ca. 1:4 ratio to IGFBP-3 binding protein.

These results show that systemic administration of IGF complex increasesadrenal tissue weight significantly, relative to body weight. It mightbe reasonably be assumed from these data that adrenal function wouldalso be affected by IGF complex administration.

The results also show that an equivalent dose of IGF-I administered inthe form of a complex with its binding protein, IGFBP-3, is superior inits effect on adrenal tissue when compared with the same dose of IGF-Ialone.

Example 2

18 healthy male and female volunteers (ages 20-53) were treated withrhIGF-I/IGFBP-3 at doses of 0, 0.3, 1.0 or 3.0 mg/kg administered dailyby intravenous infusion (15 minute infusion). rhIGF-I/IGFBP-3(manufactured as described in Sommer et al., supra) dissolved in 50 mMsodium acetate, 105 mM sodium chloride, pH 5.5, was diluted with normal(0.9%) saline. Serum samples taken at various times before, during andafter treatment were assayed for levels of various endocrine factors. Toestablish baseline values, samples taken just prior to the first dose,or a day earlier, were assayed. Samples taken on day 6 or 7 (in eachcase, 24 hours after the previous dose of rhIGF-I/IGFBP-3 had beenadministered) were used in the assays for comparison, and expressed as apercentage of baseline. In this way, the circulating levels of variousendocrine substances were determined for each patient at the beginningand at (or near) the end of the rhIGF-I/IGFBP-3 treatment regimen inorder to assess the effect of IGF-I complex administration.

Assays were performed using commercial kits according to eachmanufacturer's instructions: Nichols Institute Diagnostics, San JuanCapistrano, Calif. (DHEAS, Cortisol, LH, Erythropoietin, Calcitonin,Intact parathyroid hormone (PTH), active Renin, Total T4, Free T4,thyroglobulin, thyroid stimulating hormone(TSH)); Diagnostics SystemsLaboratories, Webster, Tex. (Prolactin, IGFBP-2, aldosterone,androstenedione, total T3, testosterone, estradiol); BiotecxLaboratories, Inc., Houston, Tex. (thyroid binding globulin (TBG)).

In addition, some assays (IGF-I, IGF-II, IGFBP-3, CBG, SHBG, DHEA,Osteocalcin, Procollagen Peptides, Bone Alkaline Phosphatase) wereperformed under contract by Endocrine Sciences, Calabasas Hills, Calif.

The results are summarized in Table 2.

TABLE 2 ASSAY Placebo 0.3 mg/kg 1.0 mg/kg 3.0 mg/kg Treatment * IGF-I 91.83 ± 7.78 150.25 ± 21.1  183.5 ± 43.27 202.67 ± 28.0 176.64 ± 36.8(0.0084) (0.0228) (0.0176) (0.00001) IGF-II 111.83 ± 29.8  67.25 ± 6.24 57.5 ± 6.03;  42.0 ± 6.25  56.82 ± 11.83 (0.0134) (0.0058) (0.0017)(0.0051) IGFBP-2  92.17 ± 29.43  159.0 ± 30.22 189.25 ± 60.2  167.7 ±123.9 172.36 ± 68.0 (0.012) (0.0404) (0.4042) (0.0043) IGFBP-3  98.0 ±13.16 111.25 ± 18.4  91.0 ± 6.38 123.67 ± 26.5 107.27 ± 21.2 (0.2688)(0.2968) (0.2289) (0.2842) DHEA  121.5 ± 39.91  89.25 ± 20.74  115.0 ±33.64 115.67 ± 34.4 105.82 ± 29.6 (0.13470) (0.7889) (0.8297) (0.4229)DHEAS  83.2 ± 17.31  114.0 ± 13.14  110.5 ± 14.18  120.0 ± 1.0 114.36 ±11.3 (0.019) (0.0355) (0.0088) (0.0116) Total T4  95.6 ± 7.06 110.75 ±12.5 107.75 ± 11.2 102.67 ± 17.8 107.46 ± 12.6 (0.0893) (0.1187)(0.5685) (0.0321) Free T4  95.67 ± 17.1  93.25 ± 18.52 101.75 ± 14.1 113.0 ± 9.54 101.73 ± 15.7 (0.8416) (0.5581) (0.0942) (0.4897) Total T3 95.67 ± 20.02  86.0 ± 6.58  91.75 ± 5.19  99.0 ± 9.17  91.64 ± 8.18(0.3117) (0.664) (0.7421) (0.6537) Androst.¹  9.83 ± 3.97  21.25 ± 14.66 22.75 ± 10.01  23.33 ± 4.73  22.36 ± 9.99 (0.2176) (0.0764) (0.0175)(0.0025) Estradiol  73.4 ± 19.6 179.25 ± 99.3 108.25 ± 38.5  113.0 ±11.36 135.36 ± 68.2  0.0121  0.0170  0.011  0.016 Prolactin 103.83 ±29.2 101.25 ± 24.2 111.25 ± 11.4  114.0 ± 33.29 108.36 ± 21.7 (0.8833)(0.5923) (0.6787) (0.7476) LH  69.83 ± 44.84  82.25 ± 57.41 118.75 ±47.8 101.33 ± 14.1  97.0 ± 47.24 (0.7291) (0.153) (0.5518) (0.2743)Cortisol  113.2 ± 40.9 104.75 ± 42.5  96.25 ± 21.7 115.67 ± 11.2 104.64± 27.8 (0.7725) (0.4545) (0.9039) (0.6858) CBG²  97.4 ± 13.5  96.0 ±7.83  101.0 ± 9.02 103.67 ± 13.9  99.91 ± 9.6 (0.8517) (0.6477) (0.5641)(0.7209) Aldosterone  45.33 ± 16.16  23.0 ± 6.58  37.75 ± 29.18  40.33 ±11.24  33.09 ± 18.94 (0.0189) (0.6591) (0.6091) (0.1861) SHBG³  104.4 ±20.45  88.75 ± 5.8 100.75 ± 14.9 127.33 ± 35.8 103.64 ± 24.4 (0.1665)(0.7658) (0.39) (0.9495) EPO⁴  163.0 ± 49.23  220.0 ± 51.02  202.0 ±86.46 153.33 ± 10.5 195.27 ± 61.9 (0.1267) (0.4561) (0.6618) (0.2611)Osteocalcin  86.5 ± 51.74 159.75 ± 94.0  71.5 ± 24.91 125.67 ± 4.16118.36 ± 66.5 (0.2244) (0.5587) (0.1233) (0.2939) Bone Ap⁵  99.0 ± 16.33 106.5 ± 11.27  103.5 ± 8.1  100.0 ± 8.19 103.64 ± 8.86 (0.4155)(0.5807) (0.9061) (0.5403) Procoll.⁶  85.33 ± 24.28 105.75 ± 9.14  114.0± 14.9  113.0 ± 17.35 110.73 ± 13.0 (0.1046) (0.0496) (0.1001) (0.0508)Active Renin  138.5 ± 68.99 123.25 ± 77.5  130.0 ± 37.5 109.33 ± 24.1121.91 ± 49.1 (0.761) (0.8081) (0.3852) (0.6165) * values computed for acombined grouping of all three rhIGF-I/IGFBP-3 treatment groups¹Androstenedione ²Cortisol Binding Globulin ³Steroid Hormone BindingGlobulin ⁴Erythropoietin ⁵Bone Alkaline Phosphatase ⁶Procollagen peptide

Measurements obtained at the end of the study were expressed as apercentage of baseline levels in each patient prior to the firstinjection of IGF-I complex (rhIGF-I/IGFBP-3). An average (±SD) of thesepercentage values were then computed for each group. P values are shownin parentheses, and were calculated as a two-tailed, unpaired t-testcomputed versus placebo.

The results clearly show that, in healthy volunteers, systemicadministration of IGF-I complex results in significantly elevated levelsof IGF-I, IGFBP-2, DHEAS. total T4, estradiol, androstenedione andprocoliagen peptide. Simultaneously, IGF-II levels are significantlylowered.

Significant elevation of DHEAS, estradiol and androstenedione levels(relative to placebo) occurs despite a reduction in aldosterone levels.DHEA, cortisol and CBG levels are not significantly changed in thisstudy. Thus the change in circulating levels of adrenal steroids isquite specific and suggests a selective stimulation of cytochromePA450c17 lyase (versus hydroxylase) activity. This effect of IGF-I invivo has not been previously demonstrated.

An increase in cytochrome b5 levels or activity might be responsible forthe increase in P450c17 lyase activity. After observing the effects ofIGF-I complex on DHEAS levels, the 5′ region of the cytochrome b5 genewas closely inspected. A portion of the 5′ region of the cytochrome b5gene was identified as containing a sequence that has similarity to anIGF-responsive element found in \

\78-450 scc, d1 crystalline and thyroglobulin genes. This potentialIGF-responsive element in cytochrome b5 gene has not been previouslydescribed.

Total T4 were also elevated significantly by IGF-I complexadministration in human subjects, while no significant changes wereobserved in free T4, total T3, TSH, or thyroglobulin levels. Sucheffects of IGF/IGFBP-3 complex in vivo have not been previouslydescribed.

The elevation of IGFBP-2 levels in response to IGF-I complexadministration was also unknown. Also unknown is the effect of changingthe ratio of circulating IGF-I to IGF-II, as accomplished in this study.IGFBP-3 levels were not significantly altered, however.

Circulating levels of procollagen peptide indicate the rate of collagendeposition in the tissues. Systemic administration of IGF-I complexelevates the level of this peptide significantly, suggesting a moreanabolic state with respect to the formation of bone and other tissue.Other markers, including markers of bone turnover, were notsignificantly different between treatment and controls in this study.

Example 3

12 females, ages 55-70, were treated with rhIGF-I/IGFBP-3 complex(manufactured as described in Sommer et al., supra) by continuoussubcutaneous infusion for seven days. Each dose group (placebo control,0.5, 1.0 and 2.0 mg/kg/day rhIGF-I/IGFBP-3 complex) consisted of threesubjects. rhIGF-I/IGFBP-3 was dissolved in 50 mM sodium acetate, 105 mMsodium chloride, pH 5.5 and diluted with normal (0.9%) saline. Bloodsamples were obtained before and following treatment.

Endocrine assays were performed as described in Example 2. Compared toplacebo, statistically significant changes were observed in the levelsof IGF-I, IGF-II, IGFBP-2, IGFBP-3, total T3 and TSH. Increases in totalT4, DHEAS and procollagen peptide were also observed.

Example 4

Patients suffering from fibromyalgia are treated in a double-blind,placebo-controlled trial. Patients meeting the American College ofRheumatology criteria for fibromyalgia are randomly assigned intoplacebo and drug groups. Patients are assessed for the severity of theirfibromyalgia prior to initiation of treatment, establishing a baseline.rhIGF-I/IGFBP-3 complex or placebo is administered by continuoussubcutaneous infusion using a portable mini-pump. rhIGF-I/IGFBP-3complex is administered for four or eight weeks.

Patients are assessed for the severity of their fibromyalgia symptoms atleast once every two weeks following initiation of treatment.Assessments include patient reported pain and fatigue using a visualranking scale, patient reported sleep quality, and other measures offibromyalgia symptoms such as assessments of mood by the AIMS or Beckdepression scales. rhIGF-I/IGFBP-3 complex reduces or alleviates thesymptoms of fibromyalgia in drug-treated patients as compared toplacebo-controlled patients.

Example 5

12 patients were treated with placebo or IGF-I/IGFBP-3 complex bycontinuous I.V. infusion for seven days (the same patients as in Example3, above). Whole blood samples were collected prior to treatment (Day 1)and immediately following the end of the infusion (Day 8). RNA wasextracted from whole blood samples as follows: Approximately 300 μl ofblood was chipped out while frozen and thawed in 1 ml of denaturingsolution from the Micro RNA Isolation Kit (Stratagene, cat#200344). Thekit protocol was modified as follows: The cells were disrupted bypassing the solution through a 22 gauge syringe needle 4 times. Thephenol:chloroform extraction step was repeated twice. Afterprecipitation with isopropyl alcohol, the pellet was resuspended in 100μL RNAse-free water and 1 μL of DNAse was added and incubated at 37° C.for 15 minutes. Then another phenol:chloroform extraction and anotherprecipitation. The final pellet was washed with 70% ETOH and dried. Itwas resuspended in 50 μL RNAse-free water. These RNAs were thawed and 20μL was brought up to 33 μL with RNAse-free water for the cDNA reactionswith the ‘Ready-To-Go’ T-Primed First-Strand Kit (Pharmacia, Cat.#27-9263-01). For PCR, 2-5 μL of the cDNAs were used per reaction alongwith 1 μL each of the primers (b5 sense: 5′- . . . CCT GCA CCA CA AGGTGT ACG ATT . . . 3′; and b5 anti sense: 5′ . . . TCC TCT GGC CAT GTATAG GCG ATA C . . . 3′) 10×PCR Buffer, 0.8 ul dNTPs, 0.5 μL Amplitaq(Perkin-Elmer Corp., Norwalk, Conn.) and brought up to 100 μL with dH20.These were run for 35-40 cycles on a thermocycler. The cycle profileused was: 95° C., 1 minute, 55° C., 30 minutes, 72° C. 30 minutes.

PCR products were analyzed by electrophoresis in agarose gels, followedby staining with ethidium bromide. PCR products were quantified bydensitometric scanning of the stained gels. The results are shown inTable 3. Three of nine IGF-I complex-treated individuals, but none ofthree placebo-treated individuals, showed at least a 10-fold increase inb5 cytochrome PCR product from Day 8 samples as compared to Day 1samples. For these three individuals, and the placebo controls, theexperiment was performed at least twice for confirmation. Allindividuals (18 samples) amplified a control product using plateletfactor 4 primers with comparable efficiency.

TABLE 3 Day 1 Day 8 Ratio (area) (area) (D8/D1) Placebo-treatedcontrols: Patient #008  4290  8661 2.02 Patient #111  7981 10680 1.34Patient #202  6455 10003 1.55 IGF-complex- treated: Patient #005  35413622 38.48 Patient #006  1433 17531 12.23 Patient #007 17069 14562 0.85Patient #201   42 3593 85.55 Patient #203  8029 10844 1.35 Patient #204 4966 7480 1.51

The patents, patent applications, and publications cited throughout thedisclosure are incorporated herein by reference in their entirety.

The present invention has been detailed both by direct description andby example. Equivalents and modifications of the present invention willbe apparent to those skilled in the art, and are encompassed within thescope of the invention.

We claim:
 1. A method for treating or alleviating the symptoms of a moodand affect disorder in a patient comprising administering to saidpatient an effective amount of IGF.
 2. The method of claim 1 whereinsaid IGF is IGF-I.
 3. The method of claim 2 wherein the IGF isadministered in combination with IGFBP-3.
 4. The method of claim 1,wherein said mood and affect disorder is selected from the groupconsisting of major depression, minor depression, cyclothymic disorder,disthymic disorder, and bipolar disorder.